Recent advances suggest that cholesterol and sphingolipids, components of membrane lipid raft domains, play critical roles in both presynaptic function and neurodegenerative disorders. We propose to investigate the roles of sphingolipid metabolizing enzymes/transporters in the coincident maintenance of neurotransmission and neuronal viability. In the last funding cycle, we identified the Drosophila slug-a-bed (slab) gene, which encodes the ceramidase enzyme at the heart of sphingolipid metabolism. SLAB ceramidase facilitates vesicular trafficking and fusion mediating neurotransmitter release. We also established a Drosophila model of Niemann Pick C (NPC) disease, a lipid-storage neurodegenerative disorder characterized by mistrafficking and accumulation of sphingolipids and cholesterol. 95% of human NPC cases are caused by mutation of the NPC1 gene, which encodes a putative sphingolipid transporter in endosomal-ike organelles. Two Drosophila NPC1 proteins, dNPC1a/b, similarly reside in presynaptic organelles and are independently essential. dNPC1 mutants impair vesicular and protein trafficking in the presynaptic terminal and cause age-progressive neurodegeneration. Thus, the hypothesis driving this proposal is that maintenance of sphingolipid domains is essential for protein and vesicle trafficking underlying presynaptic function, and that disruption of this pathway triggers synaptic dysfunction causative to neurodegeneration. We propose four Specific Aims to test this hypothesis. First, to use confocal imaging and subcellular fractionation to investigate the role of SLAB ceramidase and dNPC1a/b in lipid/protein trafficking regulation in neurons. Second, to use electrophysiology, dye imaging and electron microscopy to assay roles of sphingolipid turnover in regulating presynaptic function. Third, to use clonal techniques to generate SLAB ceramidase and dNPC1a/b deficient populations of neurons to assay the consequence on cell viability and the progression of neurodegeneration. Finally, to employ genetic and molecular interaction studies to probe the molecular mechanisms by which these proteins mediate presynaptic function or, in their absence, cause neuronal death. Our approach uniquely combines sphingolipid-pathway mutants, sophisticated cell biological approaches and Drosophila genetics to probe the emerging role of sphingolipid-dependent pathways in neuronal function. The proposed work promises to substantially increase our understanding of protein and vesicle trafficking mechanisms critical to neurotransmission, and to reveal defects of common causality to a number of disparate neurodegenerative diseases.